DREADD Ligands

DREADD (Designer Receptors Exclusively Activated by Designer Drugs) are genetically modified G protein-coupled receptors (GPCRs) that are activated by physiologically inert designer synthetic ligands (designer drugs), known as DREADD ligands. They are a chemogenetic tool, commonly used to manipulate neuronal activity and to investigate GPCR signaling pathways.

Products
Background
Literature (4)

Activators

Cat. No. Product Name / Activity
4936 Clozapine N-oxide
Activator of hM3Dq and hM4Di DREADDs
6329 Clozapine N-oxide dihydrochloride
Activator of hM3Dq and hM4Di DREADDs; water soluble version of Clozapine N-oxide (Cat. No. 4936)
7193 Deschloroclozapine
High affinity and highly potent activator of hM3Dq and hM4Di DREADDs; rapidly blood brain barrier penetrant
5548 DREADD agonist 21
Potent hM3Dq and hM4Di DREADD agonist; blood brain barrier penetrant
6422 DREADD agonist 21 dihydrochloride
Potent hM3Dq and hM4Di DREADD agonist; water soluble version of DREADD agonist 21 (Cat. No. 5548)
7197 JHU 37152
High affinity and highly potent hM3Dq and hM4Di DREADD agonist; blood brain barrier penetrant
7198 JHU 37160
High affinity and highly potent hM3Dq and hM4Di DREADD agonist; blood brain barrier penetrant
4349 Olanzapine
Highly potent hM4Di activator; also 5-HT2A and D2 antagonist
5549 Perlapine
Potent hM3Dq and hM4Di DREADD agonist in vitro
5611 Salvinorin B
Activator of κ-opioid DREADD (KORD)
7119 Sorbic acid
Activator of hFFA2-DREADDs

DREADD Mechanism of Action

The first DREADDs to be developed were derived from human muscarinic acetylcholine receptors (mAChRs) and termed hM1-5. These receptors have mutations in their orthosteric binding site, abolishing their affinity for the endogenous ligand ACh, while rendering them responsive to small molecule DREADD ligands, such as Clozapine-N-oxide (CNO, Cat. No. 4936) or Deschloroclozapine (DCZ, Cat. No. 7193).

The effect of ligand binding depends on the G protein-coupling of the DREADD. Binding of a ligand to DREADDs coupled to Gq signaling pathway (hM1Dq, hM3Dq and hM5Dq) activates phospholipase C, which catalyzes the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). Both IP3 and DAG have second messenger functions: IP3 binds to its intracellular receptors to elicit Ca2+ release from intracellular stores, while DAG activates multiple forms of protein kinase C (PKC). When activated by ligand binding, DREADDs coupled to the Gi signaling pathways cause inhibition of adenylyl cyclase (AC), leading to a decrease in intracellular cAMP levels. cAMP activates protein kinase A (PKA) and EPAC, therefore ligand binding at Gi DREADDs inhibits PKA and EPAC downstream signaling (Figure 1). Gi-coupled DREADDs are also known as inhibitory DREADDs (iDREADDs).

Following the development of muscarinic DREADDs, another iDREADD has been developed from the κ-opioid receptor, termed KORD. KORD is activated by the pharmacologically inert small molecule Salvinorin B (Cat. No. 5611), and like hM4Di, is coupled to the Gi signaling pathway. The simultaneous use of KORD and hM3Dq with their associated DREADD ligands enables researchers to have bidirectional control over neuronal activity, and the simultaneous interrogation of κ-opioid and mAChR signaling.

Signaling pathways for DREADD Ligands DREADD agonist 21, CNO and Salvinorin B.

Figure 1: Binding of DREADD ligands to Gq-DREADDs provokes neuronal firing, whereas binding to Gi-DREADDs results in inhibition of neuronal activity. Clozapine N-oxide and DREADD agonist 21 are non-selective muscarinic DREADD agonists and so can activate or inhibit neuronal activity, depending on the specific receptor being expressed. Salvinorin B is selective for the KORD receptor, which is coupled to Gi signaling; consequently binding results in inhibition of neuronal activity.

In addition to coupling to G proteins, GPCRs can also activate β-arrestin mediated signaling pathways. This non-canonical pathway can be controlled by CNO acting at a mutated M3 muscarinic receptor known as Rq(R165L), which activates arrestin signaling pathways without changes to G protein-mediated pathways.

More recently a DREADD derived from the human free fatty acid receptor 2 (FFA2) has been developed. Baring mutations that alter the length of free fatty acid that acts at the receptor, FFA2-DREADD responds to Sorbic acid (Cat. No. 7119), but not short chain free fatty acids. Using FFA-DREADD knockin mice researchers have been able to investigate the role of FFA2 in the gut.

DREADD Ligand Evolution and CNO Alternatives

CNO is a metabolite of Clozapine (Cat. No. 0444); research suggests that this metabolism is bidirectional, and that CNO may undergo reverse metabolism to clozapine in vivo in mice, rats, guinea pigs and non-human primates. Clozapine is an atypical antipsychotic that displays antagonism at dopamine receptors and 5-HT2A/2C receptors, so its presence in in vivo investigations of neuronal pathways may confound experimental results. Additionally, CNO is a substrate for the P-glycoprotein (P-gp) multidrug transporter, which is expressed on the blood brain barrier where it helps maintain the integrity of the blood brain barrier, and prevents the entry of drugs and removes them from the CNS.

The potential generation of clozapine and efflux of CNO from the brain has necessitated the development of DREADD ligand alternatives to CNO. DREADD agonist 21 (Cat. No. 6422) is a potent hM3Dq and hM4Di DREADD agonist that displays excellent blood brain barrier penetration. Similarly, Deschloroclozapine (DCZ, Cat. No. 7193) is a high affinity and highly potent activator hM3Dq and hM4Di DREADDs, which displays low 'off-target' binding to other GCPRs, ion channels and transporters. DCZ is particularly suited to in vivo use as it displays 100-fold greater affinity for hM3Dq and hM4Di compared to CNO, and exhibits rapid (approximately 10 minutes after intraperitoneal injection) binding and activation of hM3Dq and hM4Di in mice and non-human primates.

Literature for DREADD Ligands

Tocris offers the following scientific literature for DREADD Ligands to showcase our products. We invite you to request* your copy today!

*Please note that Tocris will only send literature to established scientific business / institute addresses.


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Chemogenetics Research Bulletin

Produced by Tocris, the chemogenetics research bulletin provides an introduction to chemogenetic methods to manipulate neuronal activity. It outlines the development of RASSLs, DREADDs and PSAMs, and the use of chemogenetic compounds. DREADD ligands and PSEMs available from Tocris are highlighted.

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